Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y2 receptor signaling
Retinal hypoxia is a major condition of the chronic inflammatory disease age-related macular degeneration. Extracellular ATP is a danger signal which is known to activate the NLRP3 inflammasome in various cell systems. We investigated in cultured human retinal pigment epithelial (RPE) cells whether hypoxia alters the expression of inflammasome-associated genes and whether purinergic receptor signaling contributes to the hypoxic expression of key inflammatory (NLRP3) and angiogenic factor (VEGF) genes. Hypoxia and chemical hypoxia were induced by a 0.2%-O2 atmosphere and addition of CoCl2, respectively. Gene expression was determined with real-time RT-PCR. Cytosolic NLRP3 and (pro-) IL-1β levels, and the extracellular VEGF level, were evaluated with Western blot and ELISA analyses. Cell culture in 0.2% O2 induced expression of NLRP3 and pro-IL-1β genes but not of the pro-IL-18 gene. Hypoxia also increased the cytosolic levels of NLRP3 and (pro-) IL-1β proteins. Inflammasome activation by lysosomal destabilization decreased the cell viability under hypoxic, but not control conditions. In addition to activation of IL-1 receptors, purinergic receptor signaling mediated by a pannexin-dependent release of ATP and a release of adenosine, and activation of P2Y2 and adenosine A1 receptors, was required for the full hypoxic expression of the NLRP3 gene. P2Y2 (but not A1) receptor signaling also contributed to the hypoxic expression and secretion of VEGF. The data indicate that hypoxia induces priming and activation of the NLRP3 inflammasome in cultured RPE cells. The hypoxic NLRP3 and VEGF gene expression and the secretion of VEGF are in part mediated by P2Y2 receptor signaling.
KeywordsRetinal pigment epithelium P2Y2 NLRP3 VEGF Inflammasome Hypoxia
absent in melanoma
age-related macular degeneration
apoptosis-associated speck-like protein containing a caspase-recruitment domain
caffeic acid phenethyl ester
cAMP response element-binding protein
hypoxia-inducible transcription factor
nuclear factor of activated T cell
NLR family CARD domain-containing protein
nucleotide-binding oligomerization domain receptors-like receptor protein
retinal pigment epithelium
small interfering RNA
signal transducer and activator of transcription
vascular endothelial growth factor
The authors thank Ute Weinbrecht for excellent technical assistance.
LK, AB, and MH conceived and designed the experiments. FD, PP, and MH performed the experiments. FD, AB, and MH analyzed and interpreted the data. PW, LK, and MH supervised research personnel. FD, AB, and MH drafted the manuscript. PW and LK made critical revision of the manuscript. All authors read and approved the final manuscript.
This research was supported by grants from the Deutsche Forschungsgemeinschaft (KO 1547/7-1 to L.K.) and the Geschwister Freter Stiftung (Hannover, Germany) to P.W.
Compliance with ethical standards
Conflict of interest
Fabian Doktor declares that he has no conflict of interest.
Philipp Prager declares that he has no conflict of interest.
Peter Wiedemann declares that he has no conflict of interest.
Leon Kohen declares that he has no conflict of interest.
Andreas Bringmann declares that he has no conflict of interest.
Margrit Hollborn declares that she has no conflict of interest.
All experimental protocols in this study were approved by the Ethics Committee of the University of Leipzig (approval #745, 07/25/2011).
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